The long-term goal of the research in this proposal is to understand the processes involved in mitochondrial biogenesis. The more immediate goal is to understand gene expression in this organelle. Towards this end we will analyze the expression of a subset of mitochondrial genes, the tRNA genes. Transcripts from mitochondrial tRNA genes will be identified and characterized by molecular biological and biochemical techniques. Our specific aims are to delineate the portions of tRNA genes necessary for transcription, to define the transcripts of these genes and to dissect the events necessary for the conversion of tRNA gene transcripts to functionl tRNAs. The portions of tRNA genes that are necessary for proper transcription and RNA processing will be determined by the selection of mutations in tRNA genes. Mutant tRNA genes will be analyzed with regard to their ability to support transcription and RNA processing in vivo. Elucidation of the events necessary for the conversion of tRNA gene transcripts to functional tRNAs will be accomplished by a combination of genetics and biochemistry. These experiments will include the identification and characterization of enzymes involved in mitochondrial tRNA biosynthesis. The mechanism of action of a novel locus on yeast mitochondrial DNA that is necessary for tRNA biosynthesis will be determined. Finally, experiments will be initiated to develop a cell free system for the study of transcription of mitochondrial genes and the processing of primary transcripts in vitro. The health relatedness of this project is several fold. First, mitochondria play a central role in cellular metabolism and mitochondrial derangements have been documented in neoplastic growth, heart disease and thyroid disease. An understanding of mitochondrial biogenesis may have far-reaching implications that clearly transcend the rather basic research proposed here. Second, new insights on the complexity and diversity of the biological world have already been obtained from studying mitochondrial genes and future insights are promised in the work proposed here. Third, a combination of basic scientific investigations such as proposed here, coupled with more applied and clinical investigations has in the past, and will continue in the future, to increase our ability to make progress in solving biomedical problems.